54
[10]
ELECTRON TRANSFER COMPLEXES
teresting that the enrichment capacity of both alkali-treated submitochondrial particles '7' 18 and complex II TM is exactly one additional complement of succinate dehydrogenase. Any added excess of the enzyme remains in solution and does not become membrane bound. It has been shown that succinate dehydrogenase isolated from Rhodospirillum rubrum chromatophores, which has a molecular weight of about 85,000, can cross-interact with alkali-treated submitochondrial particles to reconstitute an efficient succinoxidase system comparable in activity to a reconstituted system in which the beef heart enzyme is used.19. 20 However, cross reconstitution of the beef heart enzyme with alkali-treated chromatophores was not as effective as the system reconstituted from the R. rubrum enzyme plus alkali-treated chromatophores. 21 This may be in part related to the fact that the beef heart succinate dehydrogenase is a larger molecule (molecular weight 97,000) than the corresponing enzyme of R. rubrum. ,7 T. P. Singer, Compr. Biochem. 14, 127 (1%6). 18 W. G. Hanstein, K. A. Davis, M. A. Ghalambor, and Y. Hatefi, Biochemistry 10, 2517 (1971). ,9 y. Hatefi, K. A. Davis, H. Baltscheffsky, M. Baltscheffsky, and B. C. Johansson, Arch. Biochem. Biophys. 152, 613 (1972). 20 K. A. Davis, Y. Hatefi, I. P. Crawford, and H. Baltscheffsky, Arch. Biochem. Biophys, 180, 459 (1977). zl y. Hatefi and K. A. Davis, unpublished observations.
[10] P r e p a r a t i o n
of Cytochrome Heart
Oxidase from Beef
B y CHARLES R. H A R T Z E L L , H E L M U T B E I N E R T , BOB F . VAN G E L D E R ,
and Tsoo E. KING
electronfr(Cytochromec3 °meqUi Cytochromeaa vaentsreUcinF°ur transport
Cytochrome c 2
" ~ C y t o c h r o m e at~3Red-./
"~2H20
Cytochrome oxidase (ferrocytochrome c:O2 oxidoreductase, EC 1.9.3.1), the terminal member of the mitochondrial electron-transport chain, is a membrane-bound, multisubunit protein containing two heme a and two copper ion oxidation-reduction centers. A complicated picture of inter- and intramolecular electron transfer is emerging whereby reduced cytochrome c donates electrons to cytochrome oxidase, which
[10]
PREPARATION OF CYTOCHROME OXIDASE
55
rapidly rearranges these electrons intramolecularly and finally reduces dioxygen to water. The described methods of solubilizing cytochrome oxidase from mitochondrial membranes were developed with several aims in mind. It has become obvious that long-term interaction of the oxidase with bile salts, used for solubilization, is detrimental to overall structural and functional integrity. These procedures seek to limit bile salt treatment to a minimal period followed immediately by ammonium sulfate fractionation. In addition, preparations with high heme a, low inactive copper, and low lipid content (that give high activity and low viscosity) are needed for electromagnetic studies where concentrated solutions are required for careful quantitation. With these purposes in mind the following procedures were developed. These preparative methods each used the rationale of a "redgreen split" (sequential fragmentation) to remove reductases (complexes I, I-III, II, II-III, and III) prior to the solubilization of cytochrome oxidase (complex IV). This approach limits the time of oxidase exposure to high bile salt concentrations and clearly limits the presence of contaminating reductases in the final product. Cytochrome oxidase preparations have been reported by Fowler e t a l . , 1 Griffiths and Wharton, 2"3 and Yonetani. 4' ~ Purification Procedures Unless otherwise specified, all manipulations are performed at 0 ° to 4 °"
Protein concentration was determined according to the method of Gornall e t al. 6 as modified by Yonetani. 4 The procedure is as follows. Sample (0.05-0.15 ml, about 5 mg of protein) was mixed with 0.1 ml of 10% (w/v) potassium deoxycholate and 0.05 ml of 30% HzO2. After incubation at room temperature for 2 rain the volume was adjusted with water to 1.0 ml, after which 4.0 ml of biuret reagent were added. The solution was heated at 80 ° for 5 rain, then the A540 ,m was measured and compared with that of the bovine serum albumin standard (E~79n m of 6.67%/cm). r Ammonium sulfate was obtained either from British Drug Houses and Baker Chemicals or "enzyme grade" from Schwarz-Mann. Saturated L. R. Fowler, S. H. Richardson, and Y. Hatefi, Bio('hinl. Biophys. Acta 96, 103 (1962). D. C. W h a r t o n and A. Tzagoloff, see this series, Vol. 10 [45]. :~ D. E. Griffiths and D. C. Wharton, J. Biol. Chem. 236, 1850 (1961). 4 T. Yonetani, J. Biol. Chem. 236, 1680 (1961). 5 T. Yonetani, see this series, Vol. 10 [59]. ~ A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177,751 (1949). 7 j. F. Foster and M. D. Sterman, J. Am. Chem. So(. 78, 3656 (1956).
56
ELECTRON TRANSFER COMPLEXES
[10]
ammonium sulfate solutions (4.1 M at 25 ° and 3.9 M at 00) normally require the addition of ammonia to adjust the pH to neutrality. The pH of the solution is measured after a 10- to 100-fold dilution in water. Occasionally ammonium sulfate solutions contain large amounts of copper, so that an increased copper per heme a ratio is observed. In this situation the copper may be removed by treating saturated ammonium sulfate solutions with 0.5 g of cuprizone [bis(cyclohexanone)oxalyl dihydrazone] per liter and extracting the copper chelate with two portions of 100 ml of amyl alcohol per liter of ammonium sulfate. The ammonium sulfate is precipitated by addition of 5 volumes of ethanol. When solid ammonium sulfate is required for enzyme fractionation, the material is finely pulverized immediately prior to use. Cholic acid and deoxycholic acid (Schwarz-Mann, Sigma, CalBiochem, Koch Light, or British Drug Houses) were recrystallized as follows: nearly saturated bile acid solutions in hot ethanol (700-75 °) were treated with active charcoal (0.05 g per gram of bile acid) for 20 min. After separation of the charcoal by suction through a heated Biichner funnel (layered with 1 cm of Celite), the filtrate was cooled gradually to - 1 0 ° and crystals formed. The crystals were collected and dried at 80 ° for 1 hr. Stock solutions of the potassium or sodium salts of cholate and deoxycholate, 10% (w/v) or 20% (w/v), were stored in dark bottles at room temperature. Triton X-114 (Robin and Haas Co.) was obtained from Sigma. Variations in lots of Triton X-114 have led to small differences in both the phospholipid and heme a to protein ratios when oxidase was prepared by Procedure I. Tween 20 and Tween 80 were obtained from Sigma. Emasol 1130 was obtained from Kao Soap Co., Tokyo. It is generally recommended that purified cytochrome oxidase preparations be stored as a concentrated solution at low temperature, preferably at liquid-nitrogen temperature. These solutions should be stored in small containers, since loss of activity results from repeated freezethawing cycles. Small pellets of frozen oxidase can be made by dripping a concentrated solution from a pipette or stirring rod into liquid nitrogen. The concentration of oxidase per pellet can be determined, and only the required number of oxidase pellets need be thawed to prepare a solution. P r o c e d u r e 18
This method was described by Hartzell and Beinert 8 from procedures previously applied by Sun et al., 9 Fowler et al.,a and Yonetani 4 for cytochrome oxidase preparations. C. R. Hartzell and H. Beinert, Biochim. Biophys. Acta 368, 318 (1974). 9 F. F. Sun, K. S. Prezbindowsky, F. L. Crane, and E. E. Jacobs, Biochim. Biophys. Acta 153, 804 (1968).
[10]
PREPARATION OF CYTOCHROME OXIDASE
57
Step 1. Preparation of Starting Material. Mitochondrial paste in 0.25 M sucrose, l0 mM Tris-chloride, pH 7.4, obtained as described 1° was diluted by one-half with the same medium and either used directly or stored frozen at - 2 0 °. Step 2. Extraction of Cytochrome c Reductase Complexes (I-III and II-IH). The fresh or frozen paste was diluted by one-half again with a solution containing 1 mM histidine'HCl, 10 mM disodium dihydrogen ethylenediaminetetraacetate (EDTA), and 20 mM Tris-chloride, pH 7.4. Potassium phosphate (20 mM, pH 7.4) may be substituted for Tris-chloride with no change in procedure. After dilution, the mitochondrial paste was homogenized in a Waring blender alternating between low and high speed, using 30-sec bursts on each speed for a total of 150 sec. The protein concentration was determined by the biuret method, and additional h i s t i d i n e - E D T A - T r i s medium was added, if necessary, to yield a total protein concentration of 40 mg/ml. Triton X-114 (2.5 mg per milligram of protein) was added either neat or as a 20% dispersion in water, while the homogenized mitochondria solution was vigorously stirred. Solid KC1 to give a final concentration of 0.2 M KCI was then added. The mixture was stirred at 4 ° for 1 hr, followed by centrifugation at 2 °, either in a Beckman 30 rotor at 78,000 g for 100 min or in a Sorvall GSA rotor at 10,000 g for 10 hr. The supernatant was discarded, and the pellets were resuspended in cold h i s t i d i n e - E D T A - T r i s medium, and homogenized with a glass-Teflon homogenizer. The volume was adjusted with h i s t i d i n e - E D T A - T r i s medium to equal the original volume of mitochondrial paste, and cold 95% ethanol was added to a final concentration of 10% (v/v). The mixture was stirred for 1 hr at 4 ° or less. The solution was then centrifuged in a Sorvall GSA rotor at 27,000 g for 1 hr at 2 °. The supernatant usually is dark yellow and contains a considerable amount of Triton X-114. The pellet was resuspended in h i s t i d i n e - E D T A Tris medium with the aid of a glass-Teflon homogenizer. Freezing and storing the homogenized material in liquid N2 at this stage is recommended, as it appears to help considerably in removing additional lipid and Triton from the preparation and in effecting solubilization.
Step 3. Solubilization of Cytochrome Oxidase. The suspended crude preparation of c y t o c h r o m e oxidase was thawed, if necessary, and the protein concentration was determined. The homogenate was diluted to 40 mg of protein per milliliter with h i s t i d i n e - E D T A - T r i s medium. A quantity of a 20% (w/v) potassium cholate solution was then added sufficient to bring the cholate concentration to 1.5 mg per milligram of protein. Solid ammonium sulfate was added to 10% saturation at 4 °. The mixture was stirred 1.5 hr at 4 ° and then centrifuged in a Sorvall GSA 10 p. V. B l a i r , see this s e r i e s , Vol. 10 [12].
58
ELECTRON TRANSFER COMPLEXES
[10]
rotor at 27,000 g for 45 min. The residue was discarded. The supernatant was brought to 25% saturation with respect to ammonium sulfate, stirred 15 min at 4 ° and centrifuged at 27,000 g for 30 min. The grayish pink residue was again discarded, and the solution was adjusted to 40% saturation in ammonium sulfate by addition of a saturated neutralized-ammonium sulfate solution (3.9 M at 0 °) (sometimes it is necessary to adjust to 48% saturation in ammonium sulfate to achieve complete precipitation of cytochrome oxidase). After centrifugation at 27,000 g in the Sorvall GSA rotor for 30 min the pellet was resuspended in Tris-EDTA solution (as histidine-EDTA-Tris medium, omitting histidine).
Step 4. Fractionation with Ammonium SulJate. Additional cholate and Triton X-114 may be removed by repeating the ammonium sulfate fractionation between 25% and 39% saturation. Excessive fractionation without addition of further detergent, such as Tween 20, Tween 80, Emasol 1130, and Emasol 4130, may yield an insoluble preparation that can be resolubilized by repeating step 3 involving potassium cholate and 10% saturated ammonium sulfate. In this case further fractionation is required to remove excess cholate added the second time. Therefore, only one or at most two fractionations after the initial fractionation at 40% or 48% saturation in ammonium sulfate have been employed. P r o c e d u r e H 11
This method was developed in the laboratory of Tsoo E. King 11 using the rationale of "sequential fragmentation of the respiratory chain." Step 3 and onward follows the basic procedure of Yonetani. 4
Step 1. Preparation of Starting Material. Keilin-Hartree particle preparations from bovine heart (any method listed by King 12 may be used) are the preferred material, since these particles are enriched in respiratory-chain components. Heart mince is not a suitable starting material. Step 2. Extraction of Cytochrome c Reductase Complexes (1-III and H-Ill). To 400 ml of the heart muscle preparation of about 20 mg of protein per milliliter in 0.1 M borate-phosphate buffer, pH 7.6, are added 44 ml of 10% (w/v) sodium cholate (used as supplied by Schwarz-Mann) to give a final concentration of 1% in terms of sodium cholate. Solid ammonium sulfate is slowly added to give 25% saturation, and the pH is adjusted to 8.0 with 1 N NaOH. The mixture is allowed to stand for 1 hr with occasional agitation, and then solid ammonium sulfate is added to lJ M. Kuboyama,F. C. Yong, and T. E. King,J. Biol. Chem. 247, 6375 (1972). ~zT. E. King, see this series, Vol. 10 [37].
[10]
PREPARATION OF CYTOCHROME OXIDASE
59
obtain 35% saturation. After standing for 10 min with occasional stirring, the suspension is centrifuged for 60 rain in an IEC Model B-60 centrifuge at 30,000 g in a rotor A-170. The clear orange-red supernatant solution is discarded. The precipitate is suspended in 0.1 M sodium phosphate buffer, pH 7.4, to a final volume of 300 ml with the aid of a PotterElvehjem homogenizer.
Step 3. Solubilization of Cytochrome Oxidase. To this homogenized suspension is added 10% (w/v) sodium cholate to a final concentration of 2%. Solid ammonium sulfate is then added slowly to 25% saturation, and the pH is adjusted to between 7.4 and 7.8. The turbidity of the suspension gradualist decreases with time as the mixture is incubated for 4-12 hr. After centrifugation for 30 rain in a Sorvall SS-34 rotor at 48,000 g, the precipitate is discarded. Solid ammonium sulfate is added to the clear, greenish brown supernatant liquid to 40% saturation. After standing for 10 min with occasional stirring, the mixture is centrifuged for 15 rain. The light-orange supernatant liquid is discarded, and the greenish brown, thick precipitate is dissolved in 100 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 1.5% cholate. Step 4. Fractionation with Ammonium Sulfate. To the clear reddish green solution, saturated neutralized-ammonium sulfate solution (25 °) is added with stirring until a slight, but definite, turbidity becomes apparent. Usually the ammonium sulfate concentration required to reach this stage is about 25% saturation. The mixture is allowed to stand for about 30 min and then centrifuged in a Sorvall centrifuge (SS-34 rotor) at 48,000 g for 20 min. The small amount of precipitate is discarded, and saturated ammonium sulfate is added to the clear supernatant liquid to obtain 37% saturation. The precipitate obtained by centrifugation is redissolved in 80 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 1.5% cholate. Step 5. Refractionation with Ammonium SulJate. To the clear solution of crude cytochrome oxidase, saturated ammonium sulfate solution is again added until a slight, but persistent, turbidity exists. After standing for about 30 min, the mixture is centrifuged (Sorvall SS-34 rotor) at 48,000 g. The small amount of precipitate is discarded and the clear supernatant is again brought to 36% saturation by addition of saturated ammonium sulfate solution. The precipitate separated by centrifugation is dissolved in 60 ml of 0.1 M sodium phosphate, pH 7.4, containing 1% Emasol-1130. The clear, reddish green solution is brought to a slight, but definite, turbidity by adding saturated ammonium sulfate solution. At this stage the slightly turbid solution is either incubated for 15-30 min or allowed to stand for 12-14 hr after adding a few milliliters of the buffer medium to reduce the turbidity of the solution.
60
ELECTRON TRANSFER COMPLEXES
[10]
At the end of the incubation period, the solution is centrifuged (Sorvall SS-34 rotor) to remove precipitated material and the ammonium sulfate fractionation step (as above) is repeated twice, but each time the ammonium sulfate concentration used to precipitate the protein is reduced by 10% in saturation. The final precipitate is dissolved in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.25% Emasol to approximately 0.5 mM heine a. Usually the solution is then applied to a column (2.5 × 25 cm) of Sephadex G-100 equilibrated with 0.1 M sodium phosphate buffer, pH 7.4 containing 0.25% Emasol to remove the residual cholate and ammonium sulfate in the preparation and the eluate is used as such. In instances where a high concentration of oxidase is desired, the preparation is dialyzed against the phosphate-Emasol buffer medium for 12-14 hr. The overall yield of enzyme is 40-80%. Loss usually occurs at the fractionation step where the solution is incubated in 0.1 M phosphate buffer-containing Emasol-ll30; however, more attention is paid to the quality of the preparation at each step rather than the total yield. The preparation thus prepared is partially deficient in lipid and can be stored at - 7 5 ° for a few months. Variations To Yield Either (a) Phospholipid-Deficient or (b) Phospholipid-Sufficient Cytochrome Oxidase. ,3 The Keilin-Hartree preparation is adjusted to a protein concentration of about 20 mg/ml with 0.1 M borate-phosphate buffer. To 1.55 liters of the heart muscle preparation, 81.6 ml of 20% (w/v) sodium cholate solution is added to give a final cholate concentration of 1%. Solid ammonium sulfate is then slowly added to give 35% saturation, and the mixture is centrifuged at 27,000 g for 90 rain. The supernatant solution is discarded. The pellet is collected and suspended, with the aid of a Potter-Elvehjem homogenizer, in 930 ml (0.6 volume of the heart muscle preparation used) of 50 mM phosphate buffer, pH 7.4. To this suspension, 5.8 ml of 20% cholate solution is added per 100 ml of suspension. After the addition of cholate, the solution is again brought to 35% saturation with solid ammonium sulfate (neglecting the residue ammonium sulfate present in the sedimented pellet). Solution pH is adjusted to pH 7.4 by the simultaneous addition of concentrated ammonium hydroxide with solid ammonium sulfate (usually 5 pJ of NH4OH per gram of ammonium sulfate). The mixture is incubated for 1 hr and then centrifuged at 27,000 g for 40 min. The supernatant solutions (PS,) are pooled, and the sedimented residues are discarded. (a) Phospholipid-deficient cytochrome oxidase: To each 100 ml of greenish supernatant solution (PS,) 4.9 g ammonium sulfate is added. ,3 C. Yu, L. Yu, and T. E. King, J. Biol. Chem. 250, 1383 (1975).
[10]
PREPARATION OF CYTOCHROME OXIDASE
61
The mixture is allowed to stand for 30 min and then is centrifuged at 27,000 g for 30 min. The pellet is dissolved in 125 ml of 1.5% cholate in 50 mM phosphate buffer for the second cycle of ammonium sulfate fractionation, which is conducted by using saturated ammonium sulfate solution. The precipitate, between 27.5 and 37.5% ammonium sulfate saturation, is collected by centrifugation and suspended in 50 mM phosphate buffer for immediate use or dissolved in 0.25 M sucrose in the same buffer for storage at -75 °. (b) Phospholipid-sufficient cytochrome oxidase: The green supernatant solution (PSi) above is brought to 70% saturation in ammonium sulfate by adding solid salt. Decrease in solution pH upon addition of ammonium sulfate is compensated by addition of concentrated ammonium hydroxide. The jellylike precipitate of the crude oxidase is stuck on the wall of the beaker and the stirring rod. It is collected by decanting the turbid solution; centrifugation at this stage is not recommended. The oxidase is then dissolved in a small volume (usually at 0.1 the total volume of PS,) of 1.5% cholate in 50 mM phosphate buffer. The solution is brought to 28% saturation with a saturated ammonium sulfate solution. The precipitate is removed by centrifugation and the supernatant solution is brought to 70% saturation with solid ammonium sulfate (pH maintained with NH4OH). The precipitate, collected again by decantation, is dissolved in 1.5% cholate in 50 mM phosphate buffer to the desired oxidase concentration. The yields for both variations are found to be about 35%.
Procedure
//114, 15
This method was developed in the laboratory of B. F. van Gelder '4' ~.5 and is largely based upon the Fowler et al.' preparation as modified by MacLennan and Tzagoloff.'6 This procedure is a large-scale method.
Step 1. Preparation o f Starting Material. 12 Fat-free mince of 15 fresh bovine hearts (about 18 kg) is washed 3-4 times with ice-cold tapwater (
[10]
ELECTRON TRANSFER COMPLEXES
teresting that the enrichment capacity of both alkali-treated submitochondrial particles '7' 18 and complex II TM is exactly one additional complement of succinate dehydrogenase. Any added excess of the enzyme remains in solution and does not become membrane bound. It has been shown that succinate dehydrogenase isolated from Rhodospirillum rubrum chromatophores, which has a molecular weight of about 85,000, can cross-interact with alkali-treated submitochondrial particles to reconstitute an efficient succinoxidase system comparable in activity to a reconstituted system in which the beef heart enzyme is used.19. 20 However, cross reconstitution of the beef heart enzyme with alkali-treated chromatophores was not as effective as the system reconstituted from the R. rubrum enzyme plus alkali-treated chromatophores. 21 This may be in part related to the fact that the beef heart succinate dehydrogenase is a larger molecule (molecular weight 97,000) than the corresponing enzyme of R. rubrum. ,7 T. P. Singer, Compr. Biochem. 14, 127 (1%6). 18 W. G. Hanstein, K. A. Davis, M. A. Ghalambor, and Y. Hatefi, Biochemistry 10, 2517 (1971). ,9 y. Hatefi, K. A. Davis, H. Baltscheffsky, M. Baltscheffsky, and B. C. Johansson, Arch. Biochem. Biophys. 152, 613 (1972). 20 K. A. Davis, Y. Hatefi, I. P. Crawford, and H. Baltscheffsky, Arch. Biochem. Biophys, 180, 459 (1977). zl y. Hatefi and K. A. Davis, unpublished observations.
[10] P r e p a r a t i o n
of Cytochrome Heart
Oxidase from Beef
B y CHARLES R. H A R T Z E L L , H E L M U T B E I N E R T , BOB F . VAN G E L D E R ,
and Tsoo E. KING
electronfr(Cytochromec3 °meqUi Cytochromeaa vaentsreUcinF°ur transport
Cytochrome c 2
" ~ C y t o c h r o m e at~3Red-./
"~2H20
Cytochrome oxidase (ferrocytochrome c:O2 oxidoreductase, EC 1.9.3.1), the terminal member of the mitochondrial electron-transport chain, is a membrane-bound, multisubunit protein containing two heme a and two copper ion oxidation-reduction centers. A complicated picture of inter- and intramolecular electron transfer is emerging whereby reduced cytochrome c donates electrons to cytochrome oxidase, which
[10]
PREPARATION OF CYTOCHROME OXIDASE
55
rapidly rearranges these electrons intramolecularly and finally reduces dioxygen to water. The described methods of solubilizing cytochrome oxidase from mitochondrial membranes were developed with several aims in mind. It has become obvious that long-term interaction of the oxidase with bile salts, used for solubilization, is detrimental to overall structural and functional integrity. These procedures seek to limit bile salt treatment to a minimal period followed immediately by ammonium sulfate fractionation. In addition, preparations with high heme a, low inactive copper, and low lipid content (that give high activity and low viscosity) are needed for electromagnetic studies where concentrated solutions are required for careful quantitation. With these purposes in mind the following procedures were developed. These preparative methods each used the rationale of a "redgreen split" (sequential fragmentation) to remove reductases (complexes I, I-III, II, II-III, and III) prior to the solubilization of cytochrome oxidase (complex IV). This approach limits the time of oxidase exposure to high bile salt concentrations and clearly limits the presence of contaminating reductases in the final product. Cytochrome oxidase preparations have been reported by Fowler e t a l . , 1 Griffiths and Wharton, 2"3 and Yonetani. 4' ~ Purification Procedures Unless otherwise specified, all manipulations are performed at 0 ° to 4 °"
Protein concentration was determined according to the method of Gornall e t al. 6 as modified by Yonetani. 4 The procedure is as follows. Sample (0.05-0.15 ml, about 5 mg of protein) was mixed with 0.1 ml of 10% (w/v) potassium deoxycholate and 0.05 ml of 30% HzO2. After incubation at room temperature for 2 rain the volume was adjusted with water to 1.0 ml, after which 4.0 ml of biuret reagent were added. The solution was heated at 80 ° for 5 rain, then the A540 ,m was measured and compared with that of the bovine serum albumin standard (E~79n m of 6.67%/cm). r Ammonium sulfate was obtained either from British Drug Houses and Baker Chemicals or "enzyme grade" from Schwarz-Mann. Saturated L. R. Fowler, S. H. Richardson, and Y. Hatefi, Bio('hinl. Biophys. Acta 96, 103 (1962). D. C. W h a r t o n and A. Tzagoloff, see this series, Vol. 10 [45]. :~ D. E. Griffiths and D. C. Wharton, J. Biol. Chem. 236, 1850 (1961). 4 T. Yonetani, J. Biol. Chem. 236, 1680 (1961). 5 T. Yonetani, see this series, Vol. 10 [59]. ~ A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177,751 (1949). 7 j. F. Foster and M. D. Sterman, J. Am. Chem. So(. 78, 3656 (1956).
56
ELECTRON TRANSFER COMPLEXES
[10]
ammonium sulfate solutions (4.1 M at 25 ° and 3.9 M at 00) normally require the addition of ammonia to adjust the pH to neutrality. The pH of the solution is measured after a 10- to 100-fold dilution in water. Occasionally ammonium sulfate solutions contain large amounts of copper, so that an increased copper per heme a ratio is observed. In this situation the copper may be removed by treating saturated ammonium sulfate solutions with 0.5 g of cuprizone [bis(cyclohexanone)oxalyl dihydrazone] per liter and extracting the copper chelate with two portions of 100 ml of amyl alcohol per liter of ammonium sulfate. The ammonium sulfate is precipitated by addition of 5 volumes of ethanol. When solid ammonium sulfate is required for enzyme fractionation, the material is finely pulverized immediately prior to use. Cholic acid and deoxycholic acid (Schwarz-Mann, Sigma, CalBiochem, Koch Light, or British Drug Houses) were recrystallized as follows: nearly saturated bile acid solutions in hot ethanol (700-75 °) were treated with active charcoal (0.05 g per gram of bile acid) for 20 min. After separation of the charcoal by suction through a heated Biichner funnel (layered with 1 cm of Celite), the filtrate was cooled gradually to - 1 0 ° and crystals formed. The crystals were collected and dried at 80 ° for 1 hr. Stock solutions of the potassium or sodium salts of cholate and deoxycholate, 10% (w/v) or 20% (w/v), were stored in dark bottles at room temperature. Triton X-114 (Robin and Haas Co.) was obtained from Sigma. Variations in lots of Triton X-114 have led to small differences in both the phospholipid and heme a to protein ratios when oxidase was prepared by Procedure I. Tween 20 and Tween 80 were obtained from Sigma. Emasol 1130 was obtained from Kao Soap Co., Tokyo. It is generally recommended that purified cytochrome oxidase preparations be stored as a concentrated solution at low temperature, preferably at liquid-nitrogen temperature. These solutions should be stored in small containers, since loss of activity results from repeated freezethawing cycles. Small pellets of frozen oxidase can be made by dripping a concentrated solution from a pipette or stirring rod into liquid nitrogen. The concentration of oxidase per pellet can be determined, and only the required number of oxidase pellets need be thawed to prepare a solution. P r o c e d u r e 18
This method was described by Hartzell and Beinert 8 from procedures previously applied by Sun et al., 9 Fowler et al.,a and Yonetani 4 for cytochrome oxidase preparations. C. R. Hartzell and H. Beinert, Biochim. Biophys. Acta 368, 318 (1974). 9 F. F. Sun, K. S. Prezbindowsky, F. L. Crane, and E. E. Jacobs, Biochim. Biophys. Acta 153, 804 (1968).
[10]
PREPARATION OF CYTOCHROME OXIDASE
57
Step 1. Preparation of Starting Material. Mitochondrial paste in 0.25 M sucrose, l0 mM Tris-chloride, pH 7.4, obtained as described 1° was diluted by one-half with the same medium and either used directly or stored frozen at - 2 0 °. Step 2. Extraction of Cytochrome c Reductase Complexes (I-III and II-IH). The fresh or frozen paste was diluted by one-half again with a solution containing 1 mM histidine'HCl, 10 mM disodium dihydrogen ethylenediaminetetraacetate (EDTA), and 20 mM Tris-chloride, pH 7.4. Potassium phosphate (20 mM, pH 7.4) may be substituted for Tris-chloride with no change in procedure. After dilution, the mitochondrial paste was homogenized in a Waring blender alternating between low and high speed, using 30-sec bursts on each speed for a total of 150 sec. The protein concentration was determined by the biuret method, and additional h i s t i d i n e - E D T A - T r i s medium was added, if necessary, to yield a total protein concentration of 40 mg/ml. Triton X-114 (2.5 mg per milligram of protein) was added either neat or as a 20% dispersion in water, while the homogenized mitochondria solution was vigorously stirred. Solid KC1 to give a final concentration of 0.2 M KCI was then added. The mixture was stirred at 4 ° for 1 hr, followed by centrifugation at 2 °, either in a Beckman 30 rotor at 78,000 g for 100 min or in a Sorvall GSA rotor at 10,000 g for 10 hr. The supernatant was discarded, and the pellets were resuspended in cold h i s t i d i n e - E D T A - T r i s medium, and homogenized with a glass-Teflon homogenizer. The volume was adjusted with h i s t i d i n e - E D T A - T r i s medium to equal the original volume of mitochondrial paste, and cold 95% ethanol was added to a final concentration of 10% (v/v). The mixture was stirred for 1 hr at 4 ° or less. The solution was then centrifuged in a Sorvall GSA rotor at 27,000 g for 1 hr at 2 °. The supernatant usually is dark yellow and contains a considerable amount of Triton X-114. The pellet was resuspended in h i s t i d i n e - E D T A Tris medium with the aid of a glass-Teflon homogenizer. Freezing and storing the homogenized material in liquid N2 at this stage is recommended, as it appears to help considerably in removing additional lipid and Triton from the preparation and in effecting solubilization.
Step 3. Solubilization of Cytochrome Oxidase. The suspended crude preparation of c y t o c h r o m e oxidase was thawed, if necessary, and the protein concentration was determined. The homogenate was diluted to 40 mg of protein per milliliter with h i s t i d i n e - E D T A - T r i s medium. A quantity of a 20% (w/v) potassium cholate solution was then added sufficient to bring the cholate concentration to 1.5 mg per milligram of protein. Solid ammonium sulfate was added to 10% saturation at 4 °. The mixture was stirred 1.5 hr at 4 ° and then centrifuged in a Sorvall GSA 10 p. V. B l a i r , see this s e r i e s , Vol. 10 [12].
58
ELECTRON TRANSFER COMPLEXES
[10]
rotor at 27,000 g for 45 min. The residue was discarded. The supernatant was brought to 25% saturation with respect to ammonium sulfate, stirred 15 min at 4 ° and centrifuged at 27,000 g for 30 min. The grayish pink residue was again discarded, and the solution was adjusted to 40% saturation in ammonium sulfate by addition of a saturated neutralized-ammonium sulfate solution (3.9 M at 0 °) (sometimes it is necessary to adjust to 48% saturation in ammonium sulfate to achieve complete precipitation of cytochrome oxidase). After centrifugation at 27,000 g in the Sorvall GSA rotor for 30 min the pellet was resuspended in Tris-EDTA solution (as histidine-EDTA-Tris medium, omitting histidine).
Step 4. Fractionation with Ammonium SulJate. Additional cholate and Triton X-114 may be removed by repeating the ammonium sulfate fractionation between 25% and 39% saturation. Excessive fractionation without addition of further detergent, such as Tween 20, Tween 80, Emasol 1130, and Emasol 4130, may yield an insoluble preparation that can be resolubilized by repeating step 3 involving potassium cholate and 10% saturated ammonium sulfate. In this case further fractionation is required to remove excess cholate added the second time. Therefore, only one or at most two fractionations after the initial fractionation at 40% or 48% saturation in ammonium sulfate have been employed. P r o c e d u r e H 11
This method was developed in the laboratory of Tsoo E. King 11 using the rationale of "sequential fragmentation of the respiratory chain." Step 3 and onward follows the basic procedure of Yonetani. 4
Step 1. Preparation of Starting Material. Keilin-Hartree particle preparations from bovine heart (any method listed by King 12 may be used) are the preferred material, since these particles are enriched in respiratory-chain components. Heart mince is not a suitable starting material. Step 2. Extraction of Cytochrome c Reductase Complexes (1-III and H-Ill). To 400 ml of the heart muscle preparation of about 20 mg of protein per milliliter in 0.1 M borate-phosphate buffer, pH 7.6, are added 44 ml of 10% (w/v) sodium cholate (used as supplied by Schwarz-Mann) to give a final concentration of 1% in terms of sodium cholate. Solid ammonium sulfate is slowly added to give 25% saturation, and the pH is adjusted to 8.0 with 1 N NaOH. The mixture is allowed to stand for 1 hr with occasional agitation, and then solid ammonium sulfate is added to lJ M. Kuboyama,F. C. Yong, and T. E. King,J. Biol. Chem. 247, 6375 (1972). ~zT. E. King, see this series, Vol. 10 [37].
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obtain 35% saturation. After standing for 10 min with occasional stirring, the suspension is centrifuged for 60 rain in an IEC Model B-60 centrifuge at 30,000 g in a rotor A-170. The clear orange-red supernatant solution is discarded. The precipitate is suspended in 0.1 M sodium phosphate buffer, pH 7.4, to a final volume of 300 ml with the aid of a PotterElvehjem homogenizer.
Step 3. Solubilization of Cytochrome Oxidase. To this homogenized suspension is added 10% (w/v) sodium cholate to a final concentration of 2%. Solid ammonium sulfate is then added slowly to 25% saturation, and the pH is adjusted to between 7.4 and 7.8. The turbidity of the suspension gradualist decreases with time as the mixture is incubated for 4-12 hr. After centrifugation for 30 rain in a Sorvall SS-34 rotor at 48,000 g, the precipitate is discarded. Solid ammonium sulfate is added to the clear, greenish brown supernatant liquid to 40% saturation. After standing for 10 min with occasional stirring, the mixture is centrifuged for 15 rain. The light-orange supernatant liquid is discarded, and the greenish brown, thick precipitate is dissolved in 100 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 1.5% cholate. Step 4. Fractionation with Ammonium Sulfate. To the clear reddish green solution, saturated neutralized-ammonium sulfate solution (25 °) is added with stirring until a slight, but definite, turbidity becomes apparent. Usually the ammonium sulfate concentration required to reach this stage is about 25% saturation. The mixture is allowed to stand for about 30 min and then centrifuged in a Sorvall centrifuge (SS-34 rotor) at 48,000 g for 20 min. The small amount of precipitate is discarded, and saturated ammonium sulfate is added to the clear supernatant liquid to obtain 37% saturation. The precipitate obtained by centrifugation is redissolved in 80 ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 1.5% cholate. Step 5. Refractionation with Ammonium SulJate. To the clear solution of crude cytochrome oxidase, saturated ammonium sulfate solution is again added until a slight, but persistent, turbidity exists. After standing for about 30 min, the mixture is centrifuged (Sorvall SS-34 rotor) at 48,000 g. The small amount of precipitate is discarded and the clear supernatant is again brought to 36% saturation by addition of saturated ammonium sulfate solution. The precipitate separated by centrifugation is dissolved in 60 ml of 0.1 M sodium phosphate, pH 7.4, containing 1% Emasol-1130. The clear, reddish green solution is brought to a slight, but definite, turbidity by adding saturated ammonium sulfate solution. At this stage the slightly turbid solution is either incubated for 15-30 min or allowed to stand for 12-14 hr after adding a few milliliters of the buffer medium to reduce the turbidity of the solution.
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ELECTRON TRANSFER COMPLEXES
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At the end of the incubation period, the solution is centrifuged (Sorvall SS-34 rotor) to remove precipitated material and the ammonium sulfate fractionation step (as above) is repeated twice, but each time the ammonium sulfate concentration used to precipitate the protein is reduced by 10% in saturation. The final precipitate is dissolved in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.25% Emasol to approximately 0.5 mM heine a. Usually the solution is then applied to a column (2.5 × 25 cm) of Sephadex G-100 equilibrated with 0.1 M sodium phosphate buffer, pH 7.4 containing 0.25% Emasol to remove the residual cholate and ammonium sulfate in the preparation and the eluate is used as such. In instances where a high concentration of oxidase is desired, the preparation is dialyzed against the phosphate-Emasol buffer medium for 12-14 hr. The overall yield of enzyme is 40-80%. Loss usually occurs at the fractionation step where the solution is incubated in 0.1 M phosphate buffer-containing Emasol-ll30; however, more attention is paid to the quality of the preparation at each step rather than the total yield. The preparation thus prepared is partially deficient in lipid and can be stored at - 7 5 ° for a few months. Variations To Yield Either (a) Phospholipid-Deficient or (b) Phospholipid-Sufficient Cytochrome Oxidase. ,3 The Keilin-Hartree preparation is adjusted to a protein concentration of about 20 mg/ml with 0.1 M borate-phosphate buffer. To 1.55 liters of the heart muscle preparation, 81.6 ml of 20% (w/v) sodium cholate solution is added to give a final cholate concentration of 1%. Solid ammonium sulfate is then slowly added to give 35% saturation, and the mixture is centrifuged at 27,000 g for 90 rain. The supernatant solution is discarded. The pellet is collected and suspended, with the aid of a Potter-Elvehjem homogenizer, in 930 ml (0.6 volume of the heart muscle preparation used) of 50 mM phosphate buffer, pH 7.4. To this suspension, 5.8 ml of 20% cholate solution is added per 100 ml of suspension. After the addition of cholate, the solution is again brought to 35% saturation with solid ammonium sulfate (neglecting the residue ammonium sulfate present in the sedimented pellet). Solution pH is adjusted to pH 7.4 by the simultaneous addition of concentrated ammonium hydroxide with solid ammonium sulfate (usually 5 pJ of NH4OH per gram of ammonium sulfate). The mixture is incubated for 1 hr and then centrifuged at 27,000 g for 40 min. The supernatant solutions (PS,) are pooled, and the sedimented residues are discarded. (a) Phospholipid-deficient cytochrome oxidase: To each 100 ml of greenish supernatant solution (PS,) 4.9 g ammonium sulfate is added. ,3 C. Yu, L. Yu, and T. E. King, J. Biol. Chem. 250, 1383 (1975).
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The mixture is allowed to stand for 30 min and then is centrifuged at 27,000 g for 30 min. The pellet is dissolved in 125 ml of 1.5% cholate in 50 mM phosphate buffer for the second cycle of ammonium sulfate fractionation, which is conducted by using saturated ammonium sulfate solution. The precipitate, between 27.5 and 37.5% ammonium sulfate saturation, is collected by centrifugation and suspended in 50 mM phosphate buffer for immediate use or dissolved in 0.25 M sucrose in the same buffer for storage at -75 °. (b) Phospholipid-sufficient cytochrome oxidase: The green supernatant solution (PSi) above is brought to 70% saturation in ammonium sulfate by adding solid salt. Decrease in solution pH upon addition of ammonium sulfate is compensated by addition of concentrated ammonium hydroxide. The jellylike precipitate of the crude oxidase is stuck on the wall of the beaker and the stirring rod. It is collected by decanting the turbid solution; centrifugation at this stage is not recommended. The oxidase is then dissolved in a small volume (usually at 0.1 the total volume of PS,) of 1.5% cholate in 50 mM phosphate buffer. The solution is brought to 28% saturation with a saturated ammonium sulfate solution. The precipitate is removed by centrifugation and the supernatant solution is brought to 70% saturation with solid ammonium sulfate (pH maintained with NH4OH). The precipitate, collected again by decantation, is dissolved in 1.5% cholate in 50 mM phosphate buffer to the desired oxidase concentration. The yields for both variations are found to be about 35%.
Procedure
//114, 15
This method was developed in the laboratory of B. F. van Gelder '4' ~.5 and is largely based upon the Fowler et al.' preparation as modified by MacLennan and Tzagoloff.'6 This procedure is a large-scale method.
Step 1. Preparation o f Starting Material. 12 Fat-free mince of 15 fresh bovine hearts (about 18 kg) is washed 3-4 times with ice-cold tapwater (
